(C67Hi08U31)}, and the fmgment ions at m/z 1276 CM Irr a previous
paper [I],
we
repurted the iriduidal gly~
sidrss&urntheruuts uf =kiplostttgiagrandr$ura Gagmp, a fulk medic3ne in the southwest of China, frum which a new his-iridoid glyeoside, triphtuhde A tugether with methyl luganin, luganic acid and swcrusidewere isolated, In the cuntinuation of this study, we pursued the isolation artd strucEureelucidation uf glycusidic cunstituents ofthis piant* It was hupd to Gnd new biologically active cumpuunds and observe their iri$niflcanc.ein chemutaxanumy. We nuw repurt the structures uf thrr;c: new tritq@nuid sapunins, named triptoside A,B arrd C frum theruots oft.%plant.
The m&har&ic extract uf the ruuts wan separated as shuwn pm~ous~y [lJ_ Three saponins, tripluside A (df, B (5) and C (3) were obtain&l from the fratiioa?r which we= subjected to repeated column chrumatu~aphy an silica gel and reversed phase silica gel, whose yields were:U#7, 0.03 and U&l S%, rq&vely. On mineral &d hydrolysis, all of these sapunins yielded a cummun aglycuna On the basis uf %NMR spectral data and direct TLC mmparisun with the authentic smple, it ww shuwn tu by identical with aleanulic acid (7). A comparison of ‘%NMR signals due: tu aglymne mui@ies with those of repurted sapunins revealed that all the saponinslwere munadesmosides af J-Uglycosides [23. Tripfuside A was hydrolysed with mineral acid to yield Garabinuse, crhamnuse and ~xyluse as sugar compunents. The JWruf this sapunin was successfuflydetermined by the negative ion FAB mass spectrum, in which the m&War iun peak appeared at wzfa 1408 {[MI”
-peatuse]-, 1144 CM-2 pentuse]-, 1032 EM-3 pen* tuse]-, 866 [M-Rha-3 pentas&J-, 734 [M-Rha-4 pentusej-, 588[M-4pentuse-2Rhafand456[M-2 Rha - 5 pentuse] -. The 13CNMR sparum due to sugar moieties indicated the presence;uf seven monosaccharide units, Partial add hydrolysis of tripluside A with U,25M HCI dn 5W%EtUH produced five prusapugenins. Under the cunditiuns of mineral acid hydrulysis, prusapugenin A-X pravided L-arabinuse and L-rhamnose as sugar cornpunents, Its aregative iutl FAB mass spectrum exhibited a mul&%&%r iun peak 8% m/z 734 (Iw]* &&H&& 1)), and fragmeentiuas at m/z 588 [M - Rhal -, 456 fM -I&a -Araf-. Its ‘HNMR spe&rum shawed two anume& prutan s@als at S6.20 flI-5, br sh, and 4.9X [tH, d, J =:5.40 Hz), and its t3C NMR s-rat data indicati the presenczeuf a terminal ~-~*rham~upyr~usyl unit and aa innrer ~~~-~8b~upyr~~yl unit [anumeric ~bunsz S101,8, 104.63. In the ‘%NMR @ectrum of prusa= pugenin A-l, the signal uf c-2 due to arabinuse was shifted dawnfield at 6 76.1, while thg signals of C-l and C% 3 were displaced upfield at &B4,6 and 74.1, respectively (Table 1).This is in accurdanm with previous cases of 2a O-gfycusylated ~-~-arabinupyranusi~ [33, It was cQtT* firmed that the terminal rhamnuse is attached to the C-2 position of the inner arabinuse, Thus, the structure of prusapogenin A-1 is uleatlulic atid 3-U-rx+rhamnupyr~ anusyf (13 2~~-L~arabi~upyranuside(l), which is ide.& -1 to the sapunin CP2 pr&uusly isisolatedfrum Clmrtis &W&S [4] and Ake~tlaqkwlra [S-j. Prusapugenin A-2 gave L-arabinuse, r+-rhazzmuseand a-xyluse as sugar cumpunents on mineral acid hydrulysis, The mulecular formula C4eH7+Q15was cuntcluded&urn the peak at m/z 866 [MJ- inthe negative iun FAB mass sp~trum. The sugar sequent of pruspugenti A-2 WAS established by the fragmat ians at m/z 734 fM -XylJ -, 58g[M-Xyl-Rha]-,456[M-Xyl-Rha-Ara]-.On @omparisunuf the 13CNMR data uf prusapugenin A-2
3402
W.-G.
MA et (11.
RO
1 2 3 4
R -Ara( 2-I) Rha -AraC2-+1)Rt_ta(3+1)Xyl -A~a~2~l~Rha~3--rcl~Xyi~3--c_1~Rha -Am (2--c_l)Rha (3 --M)Xyl (3-+1)Rha(4-l)Xyi
5 -Ara(2-1)Rha
(3-1)Xyff3-+1)Rha(4-1)Xyl~3-+l~Xyi
6 -Ara(2-+l)Rha
(3 -1)Xyl
7
t3-+1)Rha~4-1)Xy1(3--,I)Xyll4-I)
Xyl
-1i
with those of 1, the former showed a set of additional signals of a terminal @-D-xylopyranosyl unit, and the C-3 of the rhamnose was displaced downfield at 5 83.0, whereas C-2 and C-4 were shifted upfield at 71.9 and 73.0, respectively (Table 1). From these results, prosapogenin A-2 is oleanolic acid 3-~-~-D-XylOpyranOSy1(1-+3)a-L-rhamnopyranosyl( l--+2)-a+arabinopyranoside (2), which is identical with eleutheroside K isolated from EIeutherococcus senticosus [6] and also from Ctemtis chinensis (named saponin CP3) [7], Patriniu scabinosaefoiia {named scopoletin) [83 and Sapindus detatiayi [9]. Prosapogenin A-3 afforded the same kinds of sugars as 2 under mineral acid hydrolysis. The negative ion FAB mass spectrum showed a molecular ion peak at m/z 1012 ([Mf- (Cg2H84019)f, and the fragment ions at m/z 866 [M-RhaJ-, 734 [M-Rha-Xyl)-, 588 EM-2Rha CM-2 Rha-Xyl-Ara]-, which -XYlf -9 456 suggested that the prosapogenin A-3 has an additional terminal rhamnose compared to 2. This was also supported by the consequence of partial acid hydrolysis on 2D-HI?I’LC [IO]. On comparison of the ’ 3C NMR spectrum of posapogenin A-3 with that of 2, a set of additional signals due to a terminal ix-L-rhamnopyranosyl unit was observed (Table 1). According to the glycosylation shift effect, the downfield shift (+ 5.2 ppm) of C-3 of xylose suggested that the terminal rhamnose should be attached to the C-3 position of xylose. Therefore, the structure of prosapogenin A-3 was deduced as oleanolic acid 3-G or-L-rhamnopyranosyl(l -+J)-b-D-xyfopyranosyl( I -+3)-aL-rhamnopyranosyl(1 -,2)-a-L-arabinopyranoside (3). This conclusion was also supported by means of 2D NMR analysis. Based on the ‘HI-‘H and 13C-1H CQSY spectra of the acetate (3a) of 3, the signals of protons and carbons of sugar moieties were assigned. The COLOC spectrum [I 1J showed coupling cross peaks of H-l of terminal rhamnose and C-3 of xylose, C-l of xylose and H-3 of inner rhamnose and ‘H-1 of inner rhamnose and C2 of arabinose. Thus, the interglycosidic linkages of 3a were further confirmed. Prosapogenin A-4 provided L-rhamnose, D-XylOSe and L-arabinose in a ratio of 2 : 2 : 1 as sugar components on mineral acid hydrolysis. The negative ion FAB mass spectrum gave a molecular ion peak at m/z 1144 f [Ml(C,,H,,O,,)} and fragment ions at 1012 [M-Xyl]+, 866 [M-Xyl-Rhaj-, 734 [M-Rha-2 XylJ-, 588 [M
-2 Xyl-2 Rha]-, 456 CM-2 Xyl-2 Rha-Ara]-. On comparison of the 13CNMR spectrum with that of 3, prosapogenin A-4 showed an additional terminal B-Dxylopyranosyl unit. It was deduced that this xylose was attached to the C-4 position of the terminal rhamnose of 3, because the signal of C-4 of this rhamnose in prosapogenin A-4 was displaced downfield at 884.7 (Table 1). Thus, prosapogenin A-4 is oleanolic acid 3-0-/?-Dxylopranosyl( i-+4)-a-L-rhamnopyranosyl( l-+3)-#&~xylopyranosyl( 1--+3)-#-L-rhamnopyranosylf l-+2)-a+arabinopyranoside (4). Prosapogenin A-5 gave D-XyfOSe, t-rhamnose and Larabinose in a ratio 3: 2: I as sugar components under mineral acid hydrolysis. The molecular ion peak appeared at m/z 1276 (CM]- (Cs2H100027)) and fragment ions at m/z II44 [M-Xyl]-, 1012 CM-2 Xyl)-, 866 CM-2 Xyj-Rha]-, 734 [M-Rha-3 Xyl]-, 588 [M-3 Xyl-2 rha], 456 [M -3 Xyl-2 Rha- Ara] - in the negative ion FAB mass spectrum. As compared with the chemical shifts of 4, the prosapogenin A-5 showed a set of additional signals of a terminal fi-D-xylopyranosyl unit, and the chemical shift of C-3 of xylose was shifted downfield at S 87.1 (Table 1). This suggested that the terminal xylose should be attached to the C-3 psotion of the xylose. Thus, the structure of prosapogenin A-5 was elucidated as oleanolic acid 3-(?-fl-D-xylopyranosyl (I -+3)-/?-D-xylopyranosyl (l-+4)-a+rhamnopyranosyl (I--, 3)-j&D-XylOpyranOSyl (I-+ 3)-a-L-rhamnopyranos yl (f-+2)-a+arabinopyranoside (5). Triploside A was identified through the direct comparison of its 13C NMR spectrum with that of 5. This showed that there was a set of additional signals of B-Dxylose. In its 13C NMR spectrum, the presence of a typical carbon signal which appeared at 676.0 due to the C-4 of xylose revealed that the terminal xylose was attached to the C-4 position of the xylose (Table 1). Based on the above evidence, the structure of triploside A was assigned as oleanolic acid 3-O-P-D-xylopyranosyl( 1+4)&D-xylopyranosyl( I -+3)-/3-D-xyiopyranosyl( l-+4)-01L-rhamnopyranosyl(1-+3)-~-D-xylopyranosyl(l+3)-at-rhamnopyranosyl (l-+2)-a-L-arabinopyranoside (6). Triploside B was hydrolysed with mineral acid to afford D-xylOSe, L-rhamnose and t-arabinose in a ratio of 3:2: 1. The negative ion FAB mass spectrum exhibited a molecular ion peak at m/z 1276 ( [M] - (C&H 100027) >
Triterpenoid saponins from Triplustegia grandi~ora
3403
Table 1. “C NMR chemical shifts of sugar moieties of saponins and prosapogenins in (400MHz, pyridine-d,, S-values)
c Ara- 1 2 3 4 5 Rha- 1 2 3 4 5 6 Xyi-1 2 3 4 5 Rha-1 2 3 4 5 6 Xyl-1 2 3 4 5 Xyl-1 2 3 4 5 Xyl-1 2 3 4 5
1
2
3
104.6 76.1 74.1 68.4 64.3 101.8 72.4 72.6 73.5 69.9 18.6
105.0 75.6 74.2 69.0 65.2 101.5 71.9 83.0 73.0 69.7 18.6 107.4 75.6 78.4 71.1 67.4
105.0
75.7 74.1 69.0 65.2 101.5 71.9 82.7 73.0 69.6 18.6 106.9 75.6 83.6 70.0 67.3 102.7 72.5 72.7 74.1 69.8 18.6
and fragment ions at pnfi 1144 [M - pentose] -, 1012 [M - 2 pentose] -, 866 [M - 2 pentose - Rha] -, 734 [866 - 3 pentose-Rha]-, 588 [734- 3 pentose - 2 Rha]-, 456 C588-4 pentose - 2 Rha] -. When triploside B was partially hydrolysed with dilute hydrochloric acid in ethanol, four prosapogenins were obtained, which were identical with 14, respectively, by comparison of physical and spectral data with authentic samples. Triploside B was confirmed as 5 by the direct comparison of physical and spectral data. Tripioside C was identical with 3 based on the analysis of the negative ion FAB mass spectrum, ‘H and 13CNMR spectral and physical data.
EZXPJIBIMENTAL
Mps are unmrr. Optical rotations were measured in MeOH. ‘H, 13Cand 2D NMR spectra were measured in pyridine-d, with 400 MHz instruments using TMS as int. standard. 2D HPTLC partial acid hydrolysis [lo] was carried out with silica gel
3s (in CDCI,) 103.7 73.3 70.8 67.8 62.3 97.8 68.8 74.0 72.6 67.2 17.2 101.4 70.8 77.8 70.7 62.1 98.1 69.9 71.5 72.6 66.7 17.2
4
5
6
105.0 75.8 74.1 69.0 65.2 101.5 71.8 82.6 72.9 69.5 18.6 106.9 75.7 83.0 69.8 67.3 102.2 72.7 72.1 84.7 68.1 18.6 107.1 76.2 78.7 71.0 67.4
104.9 75.7 74.0 68.9 65.1 101.3 71.7 82.4 72.8 69.4 18.3 106.1 75.1 82.8 69.6 67.2 102.0 72.5 72.0 84.1 67.8 18.3 105.7 74.9 87.1 68.9 66.7 106.7 75.6 77.9 70.8 67.2
104.3 75.1 73.5 68.3 64.5 100.8 71.2 81.9 72.3 69.1 17.7 105.6 74.5 82.2 68.8 66.7 101.5 72.0 71.4 83.6 68.3 17.7 104.9 74.4 86.4 68.7 66.1 103.2 73.2 75.1 76.0 64.1 106.2 75.0 77.3 70.3 67.2
60 F254 HPTLC plates (7.5 x 7.5 cm) (E. Merck), CHCl,MeOH-Hz0 (14: 6: 1) was used as a developing solvent in the first direction after saponin was partially hydrolysed in HCl vapour for 4 min. It took 35 min to complete hydrolysis in HCI vapour and then the chromatogram was developed with the CHCf,-MeOH-H,O (7: 3: 1, lower layer)-HOAc (9: 1) in the second direction. Next, it was coloured with anihne-@phthalic acid-n-B&H (2 : 3 : 200). Extraction and iso~ution of sap&s. Dried roots (2.95 kg) of T. gru~i~~a were extracted with MeOH and sepd to frs A-K as described previously Cl], Fr. H-l was chromatographed on a silica gel column by eluting with CHC&-M&H-Hz0 (50: 10: 1) and then was chromatographed on a reverse phase column (Lichroprep Rp8) (70% MeOH) to give 6 (1 g). Fr. I-l was chromatographed on a silica gel column by eluting with CHC13-MeOH-H,O (50: 10: 1) and then was chromatographed on a silica gel column by eluting with CHCl,-MeOH-H,O (10: 12: 1) to give 5 (0.4 g). Fr. K-4 was chromatographed on a silica gel column by eluting with CHCl,-MeOH-H,O(50: 10: 1) and was again chromatogra-
3404
W.-G. MA et al.
phed on a eommn of silica H by eluting with CHCI,-M&H-H,0 (10: 2: 1) to give 3 (2 g). Triploside A (6). An amorphous powder, mp 230-234” (dec.), [a];? - 36.43” (MeOH, c 0.549). ‘HNMR: 64.89 (IH, d, J =6.4 Hz, AraH-1), 5.17(fH,d,J=7.0 Hz, Xyl H-l), 522(2H,d, J =7.5 Hz, Xyf’ H-l and Xyl” H-l), 5.32 (lH, d, J =7.8 Hz, Xyl”’ H-1),624(1H,brs, RhaH-1),6.27(1H,brs, Rha’ H-1). “CNMR data see Table 1. 7’riploside B (5). An amorphous powder, mp 19&195” (dec.), CaXi7-34.41” (MeOH; c 0.494). ‘H NMR: 64.86 (lH, d, J =6.0Hz,AraH-l),5,12(lH,d,J==8.0Hz,XylH-l),5.17(1H,d, J=8.0Hz,Xyl’H-t),5.21(1H,d,J=8.0Hz,Xyl”H-lj,6.06(1H, s, Rha H-l), 6.07 (lH, s, Rha’ H-l). 13CNMR data see Table 1. Triploside C (3). An amorphous powder from MeOH, mp 210-215” (dec.), [c# -26.69” (MeOH, c 0.490). Negative ion FAB MS m/z 1012 ([Ml-, C,,H,,O,,), 866 CM-Rha]-, 734 [M-Rha-Xyl]-, 588 CM-2 Rha-Xyl]-, 456 CM-2 Rha-Xyi-Araf-. ‘HNMR:64.84(1H,d,J=6.0Hz,AraH-1), 5.3O(lH, d, J=7.6 Hz, Xyl H-l), 6.21 (IH, s, Rha’ H-l), 6.25 (lH, s, Rha H-l). ‘3CNMR data see Table 1. Partiar! acid hydrolysis of compounds 6 and 5. Compounds 6 (800 mg) and 5 (70 mg) were hydrolysed with 0.25 M NC1 in 50% EtOH under reflux for 20 and 50 min, respectively. The reaction mixts were neutralized with a saturated soin of NaHCO, and coned to dryness. The residues were partitioned between H,O and n-BuOH. The n-BuOH extracts were coned to dryness and the residues were chromatographed on silica gel by eluting with CHCl,-MeOH-H,O (7: 3: 1, lower layer). Compound 6 gave five prosapogenins, A-l to A-5. Compound 5 afforded four prosapogenins which were identified with A-l to A-4, respectively, as well as aglycone 7. Prosapogenin A-l (1). An amorphous powder from MeOH (15 mg), mp 225-230” (dec.) {ref. [6] 220-224” (dec.) )_ ‘H NMR: 64.91 (lH, d, J=5.4Hz, Ara H-l), 6.20 (tH, br s, Rha H-l). t3CNMR data see Table 1. Prosupogenin A-2 (2). An amorphous powder from MeOH (8 mg), mp 240-245” (dec.) {ref. [43 241-243” (dec.)). “HNMR: 54.88 (lH, d, J--5.9 Hz, Ara H-l), 5.31 (lH, d, J=7.4 Hz, Xyl H-l), 6.20 (IH, br s, Rha H-l). 13CNMR data see Table 1. Prosapogenin A-3 (3). An Amorphous powder from MeOH (9 mg), mp 207-212” (dec.). ‘H NMR: 64.86 (IH, d, J =6.1 Hz, AraH-1),5.34(1H,d,J-7.8 Hz,XylH-1),6,25(1H,s,RhaH-l), 6.29 (IH, s, Rha’ H-l). 13CNMR data see Table 1.
Prosapogenin A-4 (4). An amorphous powder from MeOH (7 mg), mp 206-210” (dec.). ‘HNMR: 64.83 (lH, d, J=6.1 Hz, Ara H-l), 5.25 (lH, d, J=7.8 Hz, Xyl’ H-l), 5.48 (IH, d, Xyl H-l), 6.17 (2H, br s, Rha’ H-l and Rha H-l). r3CNMR data see Table 1. Prosapogenin A-5 (5). An amorphous powder from MeOH (5 mg), mp 210-215” (dec.). Aglycone 7, Needles (10 mg), mp 305-308”. Acetylation ofcompound 3. Compound 3 (100 mg) was added to Ac,O-pyridine (1: 1)(1.5 ml) in a micro-tube. After standing at room temp. for 48 hr, the soln was poured into glacial H,O and then extracted with CHCl,. From the CHCl, extract, 3a was obtained. Compound 39 gave yellowish crystals from EtOH, mp 160-163”. ‘H NMR: 63.78 (lH, d, J = 8.6 Hz, Xyi H-3), 3.99 (IH, s, Rha H-3), 4.01 (lH, br s, Ara H-2), 4.46 (lH, d, J = 6.1 Hz, Ara H-lj,4.85(1H,s,Rha’H-l),4.49(1H,d,J=6.8Hz,XylH-l), 5.02 (lH, br s, Rha H-l). 13CNMR data see Table 1. Acknowledgement-We thank Dr R. Kasai (Hiroshima Wniversity, Japan) for FAB mass spectra.
REFERENCES
1. Ma, W. G., Wang, D. Z., Zeng, Y. L. and Yang, C. R. (1991) Acta Bot. Yunnan. (in press). 2. Kizu, H. and Tomimori, T. (1982) Chem, Pharm. Bull. 30,859. 3. Mizutani, K., Hayashi, A., Kasai, R. and Tanaka, 0. (1984) Carbohyd. Res. 126, 177. 4. Kizu, H. and Kawasaki, T. (1979) Chem. Pharm. Bull. 27, 2388. 5, Higuchi, R. and Kawasaki, T. (1976) Chem. Pharm, Bull. 24,
1021. 6. Froiova, G. M., Ovodov, Y. S. and Saprunov, N, f. (1971) Khim. Prir. Soedin. 7, 614. 7. Kizu, H. and Tomimori, T. (1980) Chem Pharm. Bull. 28, 2827. 8, Choi, J. S. and Woo, W, S, (1984) Arch. Pharmacol, Res. 7, 121. 9. Nakayama, K., Fujino, H., Kasai, R., Tanaka, 0. and Zhou, J. (1986) Chem, Pharm. Bull, 34 2209, 10. Heisig W. (1988) Planta Med. 54, 582. 11. Li, X. C., Wang, D. Z. and Yang, C. R. (1990) Chin. J. h4agn. Reson. 7, 261.
paper [I],
we
repurted the iriduidal gly~
sidrss&urntheruuts uf =kiplostttgiagrandr$ura Gagmp, a fulk medic3ne in the southwest of China, frum which a new his-iridoid glyeoside, triphtuhde A tugether with methyl luganin, luganic acid and swcrusidewere isolated, In the cuntinuation of this study, we pursued the isolation artd strucEureelucidation uf glycusidic cunstituents ofthis piant* It was hupd to Gnd new biologically active cumpuunds and observe their iri$niflcanc.ein chemutaxanumy. We nuw repurt the structures uf thrr;c: new tritq@nuid sapunins, named triptoside A,B arrd C frum theruots oft.%plant.
The m&har&ic extract uf the ruuts wan separated as shuwn pm~ous~y [lJ_ Three saponins, tripluside A (df, B (5) and C (3) were obtain&l from the fratiioa?r which we= subjected to repeated column chrumatu~aphy an silica gel and reversed phase silica gel, whose yields were:U#7, 0.03 and U&l S%, rq&vely. On mineral &d hydrolysis, all of these sapunins yielded a cummun aglycuna On the basis uf %NMR spectral data and direct TLC mmparisun with the authentic smple, it ww shuwn tu by identical with aleanulic acid (7). A comparison of ‘%NMR signals due: tu aglymne mui@ies with those of repurted sapunins revealed that all the saponinslwere munadesmosides af J-Uglycosides [23. Tripfuside A was hydrolysed with mineral acid to yield Garabinuse, crhamnuse and ~xyluse as sugar compunents. The JWruf this sapunin was successfuflydetermined by the negative ion FAB mass spectrum, in which the m&War iun peak appeared at wzfa 1408 {[MI”
-peatuse]-, 1144 CM-2 pentuse]-, 1032 EM-3 pen* tuse]-, 866 [M-Rha-3 pentas&J-, 734 [M-Rha-4 pentusej-, 588[M-4pentuse-2Rhafand456[M-2 Rha - 5 pentuse] -. The 13CNMR sparum due to sugar moieties indicated the presence;uf seven monosaccharide units, Partial add hydrolysis of tripluside A with U,25M HCI dn 5W%EtUH produced five prusapugenins. Under the cunditiuns of mineral acid hydrulysis, prusapugenin A-X pravided L-arabinuse and L-rhamnose as sugar cornpunents, Its aregative iutl FAB mass spectrum exhibited a mul&%&%r iun peak 8% m/z 734 (Iw]* &&H&& 1)), and fragmeentiuas at m/z 588 [M - Rhal -, 456 fM -I&a -Araf-. Its ‘HNMR spe&rum shawed two anume& prutan s@als at S6.20 flI-5, br sh, and 4.9X [tH, d, J =:5.40 Hz), and its t3C NMR s-rat data indicati the presenczeuf a terminal ~-~*rham~upyr~usyl unit and aa innrer ~~~-~8b~upyr~~yl unit [anumeric ~bunsz S101,8, 104.63. In the ‘%NMR @ectrum of prusa= pugenin A-l, the signal uf c-2 due to arabinuse was shifted dawnfield at 6 76.1, while thg signals of C-l and C% 3 were displaced upfield at &B4,6 and 74.1, respectively (Table 1).This is in accurdanm with previous cases of 2a O-gfycusylated ~-~-arabinupyranusi~ [33, It was cQtT* firmed that the terminal rhamnuse is attached to the C-2 position of the inner arabinuse, Thus, the structure of prusapogenin A-1 is uleatlulic atid 3-U-rx+rhamnupyr~ anusyf (13 2~~-L~arabi~upyranuside(l), which is ide.& -1 to the sapunin CP2 pr&uusly isisolatedfrum Clmrtis &W&S [4] and Ake~tlaqkwlra [S-j. Prusapugenin A-2 gave L-arabinuse, r+-rhazzmuseand a-xyluse as sugar cumpunents on mineral acid hydrulysis, The mulecular formula C4eH7+Q15was cuntcluded&urn the peak at m/z 866 [MJ- inthe negative iun FAB mass sp~trum. The sugar sequent of pruspugenti A-2 WAS established by the fragmat ians at m/z 734 fM -XylJ -, 58g[M-Xyl-Rha]-,456[M-Xyl-Rha-Ara]-.On @omparisunuf the 13CNMR data uf prusapugenin A-2
3402
W.-G.
MA et (11.
RO
1 2 3 4
R -Ara( 2-I) Rha -AraC2-+1)Rt_ta(3+1)Xyl -A~a~2~l~Rha~3--rcl~Xyi~3--c_1~Rha -Am (2--c_l)Rha (3 --M)Xyl (3-+1)Rha(4-l)Xyi
5 -Ara(2-1)Rha
(3-1)Xyff3-+1)Rha(4-1)Xyl~3-+l~Xyi
6 -Ara(2-+l)Rha
(3 -1)Xyl
7
t3-+1)Rha~4-1)Xy1(3--,I)Xyll4-I)
Xyl
-1i
with those of 1, the former showed a set of additional signals of a terminal @-D-xylopyranosyl unit, and the C-3 of the rhamnose was displaced downfield at 5 83.0, whereas C-2 and C-4 were shifted upfield at 71.9 and 73.0, respectively (Table 1). From these results, prosapogenin A-2 is oleanolic acid 3-~-~-D-XylOpyranOSy1(1-+3)a-L-rhamnopyranosyl( l--+2)-a+arabinopyranoside (2), which is identical with eleutheroside K isolated from EIeutherococcus senticosus [6] and also from Ctemtis chinensis (named saponin CP3) [7], Patriniu scabinosaefoiia {named scopoletin) [83 and Sapindus detatiayi [9]. Prosapogenin A-3 afforded the same kinds of sugars as 2 under mineral acid hydrolysis. The negative ion FAB mass spectrum showed a molecular ion peak at m/z 1012 ([Mf- (Cg2H84019)f, and the fragment ions at m/z 866 [M-RhaJ-, 734 [M-Rha-Xyl)-, 588 EM-2Rha CM-2 Rha-Xyl-Ara]-, which -XYlf -9 456 suggested that the prosapogenin A-3 has an additional terminal rhamnose compared to 2. This was also supported by the consequence of partial acid hydrolysis on 2D-HI?I’LC [IO]. On comparison of the ’ 3C NMR spectrum of posapogenin A-3 with that of 2, a set of additional signals due to a terminal ix-L-rhamnopyranosyl unit was observed (Table 1). According to the glycosylation shift effect, the downfield shift (+ 5.2 ppm) of C-3 of xylose suggested that the terminal rhamnose should be attached to the C-3 position of xylose. Therefore, the structure of prosapogenin A-3 was deduced as oleanolic acid 3-G or-L-rhamnopyranosyl(l -+J)-b-D-xyfopyranosyl( I -+3)-aL-rhamnopyranosyl(1 -,2)-a-L-arabinopyranoside (3). This conclusion was also supported by means of 2D NMR analysis. Based on the ‘HI-‘H and 13C-1H CQSY spectra of the acetate (3a) of 3, the signals of protons and carbons of sugar moieties were assigned. The COLOC spectrum [I 1J showed coupling cross peaks of H-l of terminal rhamnose and C-3 of xylose, C-l of xylose and H-3 of inner rhamnose and ‘H-1 of inner rhamnose and C2 of arabinose. Thus, the interglycosidic linkages of 3a were further confirmed. Prosapogenin A-4 provided L-rhamnose, D-XylOSe and L-arabinose in a ratio of 2 : 2 : 1 as sugar components on mineral acid hydrolysis. The negative ion FAB mass spectrum gave a molecular ion peak at m/z 1144 f [Ml(C,,H,,O,,)} and fragment ions at 1012 [M-Xyl]+, 866 [M-Xyl-Rhaj-, 734 [M-Rha-2 XylJ-, 588 [M
-2 Xyl-2 Rha]-, 456 CM-2 Xyl-2 Rha-Ara]-. On comparison of the 13CNMR spectrum with that of 3, prosapogenin A-4 showed an additional terminal B-Dxylopyranosyl unit. It was deduced that this xylose was attached to the C-4 position of the terminal rhamnose of 3, because the signal of C-4 of this rhamnose in prosapogenin A-4 was displaced downfield at 884.7 (Table 1). Thus, prosapogenin A-4 is oleanolic acid 3-0-/?-Dxylopranosyl( i-+4)-a-L-rhamnopyranosyl( l-+3)-#&~xylopyranosyl( 1--+3)-#-L-rhamnopyranosylf l-+2)-a+arabinopyranoside (4). Prosapogenin A-5 gave D-XyfOSe, t-rhamnose and Larabinose in a ratio 3: 2: I as sugar components under mineral acid hydrolysis. The molecular ion peak appeared at m/z 1276 (CM]- (Cs2H100027)) and fragment ions at m/z II44 [M-Xyl]-, 1012 CM-2 Xyl)-, 866 CM-2 Xyj-Rha]-, 734 [M-Rha-3 Xyl]-, 588 [M-3 Xyl-2 rha], 456 [M -3 Xyl-2 Rha- Ara] - in the negative ion FAB mass spectrum. As compared with the chemical shifts of 4, the prosapogenin A-5 showed a set of additional signals of a terminal fi-D-xylopyranosyl unit, and the chemical shift of C-3 of xylose was shifted downfield at S 87.1 (Table 1). This suggested that the terminal xylose should be attached to the C-3 psotion of the xylose. Thus, the structure of prosapogenin A-5 was elucidated as oleanolic acid 3-(?-fl-D-xylopyranosyl (I -+3)-/?-D-xylopyranosyl (l-+4)-a+rhamnopyranosyl (I--, 3)-j&D-XylOpyranOSyl (I-+ 3)-a-L-rhamnopyranos yl (f-+2)-a+arabinopyranoside (5). Triploside A was identified through the direct comparison of its 13C NMR spectrum with that of 5. This showed that there was a set of additional signals of B-Dxylose. In its 13C NMR spectrum, the presence of a typical carbon signal which appeared at 676.0 due to the C-4 of xylose revealed that the terminal xylose was attached to the C-4 position of the xylose (Table 1). Based on the above evidence, the structure of triploside A was assigned as oleanolic acid 3-O-P-D-xylopyranosyl( 1+4)&D-xylopyranosyl( I -+3)-/3-D-xyiopyranosyl( l-+4)-01L-rhamnopyranosyl(1-+3)-~-D-xylopyranosyl(l+3)-at-rhamnopyranosyl (l-+2)-a-L-arabinopyranoside (6). Triploside B was hydrolysed with mineral acid to afford D-xylOSe, L-rhamnose and t-arabinose in a ratio of 3:2: 1. The negative ion FAB mass spectrum exhibited a molecular ion peak at m/z 1276 ( [M] - (C&H 100027) >
Triterpenoid saponins from Triplustegia grandi~ora
3403
Table 1. “C NMR chemical shifts of sugar moieties of saponins and prosapogenins in (400MHz, pyridine-d,, S-values)
c Ara- 1 2 3 4 5 Rha- 1 2 3 4 5 6 Xyi-1 2 3 4 5 Rha-1 2 3 4 5 6 Xyl-1 2 3 4 5 Xyl-1 2 3 4 5 Xyl-1 2 3 4 5
1
2
3
104.6 76.1 74.1 68.4 64.3 101.8 72.4 72.6 73.5 69.9 18.6
105.0 75.6 74.2 69.0 65.2 101.5 71.9 83.0 73.0 69.7 18.6 107.4 75.6 78.4 71.1 67.4
105.0
75.7 74.1 69.0 65.2 101.5 71.9 82.7 73.0 69.6 18.6 106.9 75.6 83.6 70.0 67.3 102.7 72.5 72.7 74.1 69.8 18.6
and fragment ions at pnfi 1144 [M - pentose] -, 1012 [M - 2 pentose] -, 866 [M - 2 pentose - Rha] -, 734 [866 - 3 pentose-Rha]-, 588 [734- 3 pentose - 2 Rha]-, 456 C588-4 pentose - 2 Rha] -. When triploside B was partially hydrolysed with dilute hydrochloric acid in ethanol, four prosapogenins were obtained, which were identical with 14, respectively, by comparison of physical and spectral data with authentic samples. Triploside B was confirmed as 5 by the direct comparison of physical and spectral data. Tripioside C was identical with 3 based on the analysis of the negative ion FAB mass spectrum, ‘H and 13CNMR spectral and physical data.
EZXPJIBIMENTAL
Mps are unmrr. Optical rotations were measured in MeOH. ‘H, 13Cand 2D NMR spectra were measured in pyridine-d, with 400 MHz instruments using TMS as int. standard. 2D HPTLC partial acid hydrolysis [lo] was carried out with silica gel
3s (in CDCI,) 103.7 73.3 70.8 67.8 62.3 97.8 68.8 74.0 72.6 67.2 17.2 101.4 70.8 77.8 70.7 62.1 98.1 69.9 71.5 72.6 66.7 17.2
4
5
6
105.0 75.8 74.1 69.0 65.2 101.5 71.8 82.6 72.9 69.5 18.6 106.9 75.7 83.0 69.8 67.3 102.2 72.7 72.1 84.7 68.1 18.6 107.1 76.2 78.7 71.0 67.4
104.9 75.7 74.0 68.9 65.1 101.3 71.7 82.4 72.8 69.4 18.3 106.1 75.1 82.8 69.6 67.2 102.0 72.5 72.0 84.1 67.8 18.3 105.7 74.9 87.1 68.9 66.7 106.7 75.6 77.9 70.8 67.2
104.3 75.1 73.5 68.3 64.5 100.8 71.2 81.9 72.3 69.1 17.7 105.6 74.5 82.2 68.8 66.7 101.5 72.0 71.4 83.6 68.3 17.7 104.9 74.4 86.4 68.7 66.1 103.2 73.2 75.1 76.0 64.1 106.2 75.0 77.3 70.3 67.2
60 F254 HPTLC plates (7.5 x 7.5 cm) (E. Merck), CHCl,MeOH-Hz0 (14: 6: 1) was used as a developing solvent in the first direction after saponin was partially hydrolysed in HCl vapour for 4 min. It took 35 min to complete hydrolysis in HCI vapour and then the chromatogram was developed with the CHCf,-MeOH-H,O (7: 3: 1, lower layer)-HOAc (9: 1) in the second direction. Next, it was coloured with anihne-@phthalic acid-n-B&H (2 : 3 : 200). Extraction and iso~ution of sap&s. Dried roots (2.95 kg) of T. gru~i~~a were extracted with MeOH and sepd to frs A-K as described previously Cl], Fr. H-l was chromatographed on a silica gel column by eluting with CHC&-M&H-Hz0 (50: 10: 1) and then was chromatographed on a reverse phase column (Lichroprep Rp8) (70% MeOH) to give 6 (1 g). Fr. I-l was chromatographed on a silica gel column by eluting with CHC13-MeOH-H,O (50: 10: 1) and then was chromatographed on a silica gel column by eluting with CHCl,-MeOH-H,O (10: 12: 1) to give 5 (0.4 g). Fr. K-4 was chromatographed on a silica gel column by eluting with CHCl,-MeOH-H,O(50: 10: 1) and was again chromatogra-
3404
W.-G. MA et al.
phed on a eommn of silica H by eluting with CHCI,-M&H-H,0 (10: 2: 1) to give 3 (2 g). Triploside A (6). An amorphous powder, mp 230-234” (dec.), [a];? - 36.43” (MeOH, c 0.549). ‘HNMR: 64.89 (IH, d, J =6.4 Hz, AraH-1), 5.17(fH,d,J=7.0 Hz, Xyl H-l), 522(2H,d, J =7.5 Hz, Xyf’ H-l and Xyl” H-l), 5.32 (lH, d, J =7.8 Hz, Xyl”’ H-1),624(1H,brs, RhaH-1),6.27(1H,brs, Rha’ H-1). “CNMR data see Table 1. 7’riploside B (5). An amorphous powder, mp 19&195” (dec.), CaXi7-34.41” (MeOH; c 0.494). ‘H NMR: 64.86 (lH, d, J =6.0Hz,AraH-l),5,12(lH,d,J==8.0Hz,XylH-l),5.17(1H,d, J=8.0Hz,Xyl’H-t),5.21(1H,d,J=8.0Hz,Xyl”H-lj,6.06(1H, s, Rha H-l), 6.07 (lH, s, Rha’ H-l). 13CNMR data see Table 1. Triploside C (3). An amorphous powder from MeOH, mp 210-215” (dec.), [c# -26.69” (MeOH, c 0.490). Negative ion FAB MS m/z 1012 ([Ml-, C,,H,,O,,), 866 CM-Rha]-, 734 [M-Rha-Xyl]-, 588 CM-2 Rha-Xyl]-, 456 CM-2 Rha-Xyi-Araf-. ‘HNMR:64.84(1H,d,J=6.0Hz,AraH-1), 5.3O(lH, d, J=7.6 Hz, Xyl H-l), 6.21 (IH, s, Rha’ H-l), 6.25 (lH, s, Rha H-l). ‘3CNMR data see Table 1. Partiar! acid hydrolysis of compounds 6 and 5. Compounds 6 (800 mg) and 5 (70 mg) were hydrolysed with 0.25 M NC1 in 50% EtOH under reflux for 20 and 50 min, respectively. The reaction mixts were neutralized with a saturated soin of NaHCO, and coned to dryness. The residues were partitioned between H,O and n-BuOH. The n-BuOH extracts were coned to dryness and the residues were chromatographed on silica gel by eluting with CHCl,-MeOH-H,O (7: 3: 1, lower layer). Compound 6 gave five prosapogenins, A-l to A-5. Compound 5 afforded four prosapogenins which were identified with A-l to A-4, respectively, as well as aglycone 7. Prosapogenin A-l (1). An amorphous powder from MeOH (15 mg), mp 225-230” (dec.) {ref. [6] 220-224” (dec.) )_ ‘H NMR: 64.91 (lH, d, J=5.4Hz, Ara H-l), 6.20 (tH, br s, Rha H-l). t3CNMR data see Table 1. Prosupogenin A-2 (2). An amorphous powder from MeOH (8 mg), mp 240-245” (dec.) {ref. [43 241-243” (dec.)). “HNMR: 54.88 (lH, d, J--5.9 Hz, Ara H-l), 5.31 (lH, d, J=7.4 Hz, Xyl H-l), 6.20 (IH, br s, Rha H-l). 13CNMR data see Table 1. Prosapogenin A-3 (3). An Amorphous powder from MeOH (9 mg), mp 207-212” (dec.). ‘H NMR: 64.86 (IH, d, J =6.1 Hz, AraH-1),5.34(1H,d,J-7.8 Hz,XylH-1),6,25(1H,s,RhaH-l), 6.29 (IH, s, Rha’ H-l). 13CNMR data see Table 1.
Prosapogenin A-4 (4). An amorphous powder from MeOH (7 mg), mp 206-210” (dec.). ‘HNMR: 64.83 (lH, d, J=6.1 Hz, Ara H-l), 5.25 (lH, d, J=7.8 Hz, Xyl’ H-l), 5.48 (IH, d, Xyl H-l), 6.17 (2H, br s, Rha’ H-l and Rha H-l). r3CNMR data see Table 1. Prosapogenin A-5 (5). An amorphous powder from MeOH (5 mg), mp 210-215” (dec.). Aglycone 7, Needles (10 mg), mp 305-308”. Acetylation ofcompound 3. Compound 3 (100 mg) was added to Ac,O-pyridine (1: 1)(1.5 ml) in a micro-tube. After standing at room temp. for 48 hr, the soln was poured into glacial H,O and then extracted with CHCl,. From the CHCl, extract, 3a was obtained. Compound 39 gave yellowish crystals from EtOH, mp 160-163”. ‘H NMR: 63.78 (lH, d, J = 8.6 Hz, Xyi H-3), 3.99 (IH, s, Rha H-3), 4.01 (lH, br s, Ara H-2), 4.46 (lH, d, J = 6.1 Hz, Ara H-lj,4.85(1H,s,Rha’H-l),4.49(1H,d,J=6.8Hz,XylH-l), 5.02 (lH, br s, Rha H-l). 13CNMR data see Table 1. Acknowledgement-We thank Dr R. Kasai (Hiroshima Wniversity, Japan) for FAB mass spectra.
REFERENCES
1. Ma, W. G., Wang, D. Z., Zeng, Y. L. and Yang, C. R. (1991) Acta Bot. Yunnan. (in press). 2. Kizu, H. and Tomimori, T. (1982) Chem, Pharm. Bull. 30,859. 3. Mizutani, K., Hayashi, A., Kasai, R. and Tanaka, 0. (1984) Carbohyd. Res. 126, 177. 4. Kizu, H. and Kawasaki, T. (1979) Chem. Pharm. Bull. 27, 2388. 5, Higuchi, R. and Kawasaki, T. (1976) Chem. Pharm, Bull. 24,
1021. 6. Froiova, G. M., Ovodov, Y. S. and Saprunov, N, f. (1971) Khim. Prir. Soedin. 7, 614. 7. Kizu, H. and Tomimori, T. (1980) Chem Pharm. Bull. 28, 2827. 8, Choi, J. S. and Woo, W, S, (1984) Arch. Pharmacol, Res. 7, 121. 9. Nakayama, K., Fujino, H., Kasai, R., Tanaka, 0. and Zhou, J. (1986) Chem, Pharm. Bull, 34 2209, 10. Heisig W. (1988) Planta Med. 54, 582. 11. Li, X. C., Wang, D. Z. and Yang, C. R. (1990) Chin. J. h4agn. Reson. 7, 261.
